The overall objective of this project is to understand the cellular and molecular mechanisms responsible for the specification, patterning, and differentiation of internal organs during development. More specifically, how the elaborate network of blood vessels arises during vertebrate embryogenesis. The model system we use for these studies is the zebrafish, a genetically tractable animal with a physically accessible, optically clear embryo. These features provide a tremendous advantage for studying vascular development, because they permit direct, noninvasive observation of every blood vessel in a living embryo throughout its development and the isolation of mutants that cause defects in the formation of embryonic blood vessels. The major aims of the laboratory include:(i) Understanding the basis for the defects in three localized vascular patterning mutants that have either disruption of cranial vessel formation, formation of abnormal arterial-venous connections, or localized blockage of circulation. The mutated gene in gridlock mutants has now been shown to be a hairy-related bHLH factor expressed specifically in the dorsal aorta. The molecular identification of the mutated loci in the other two mutants is in progress.(ii) Elucidating the signals and molecular pathways directing the formation and differentiation of trunk blood vessels. We have obtained genetic, molecular, and experimental evidence demonstrating that the well-characterized Hedgehog and Notch signaling pathways have novel, previously unknown functional roles during blood vessel formation. Hedgehog signaling is necessary for specification and formation of the trunk dorsal aorta. Notch signaling, in contrast, is not required for the initial formation of blood vessels but is required for their proper arterial-venous differentiation.(iii) Developing new experimental tools for studying blood vessel formation in the zebrafish. Using a novel confocal microangiographic technique we devised, we have prepared a detailed three-dimensional atlas of the complete vascular circuitry of the zebrafish embryo and early larva. An interactive, online version of this atlas is available at: http://mgchd1.nichd.nih.gov:8000/zfatlas/Intro%20Page/intro1.html . We have also generated transgenic zebrafish lines expressing green fluorescent protein (GFP) in blood vessels, making it possible for us to visualize the dynamics of blood vessel formation in living embryos.